The ADAM (A Disintegrin And Metalloproteinase) family of mammalian zinc metalloproteinases is a recently characterized group of membrane bound proteins which have been implicated in cell adhesion and extracellular matrix proteolysis. These proteins have also been referred to as MDCs (metalloproteinase/disintegrin/cysteine-rich), cellular disintegrins, disintegrin-metalloproteinases and metalloproteinase-disintegrins. All ADAM family members display a common domain organization and are unique among cell surface proteins in possessing both a potential adhesion domain as well as a potential protease domain. Since cell surface proteolysis plays an important role in development and pathology and since so few cell-surface membrane-anchored proteases have been described, ADAMs are believed to play important roles in development and disease (Stone, et al., J. Protein Chem., 1999, 18, 447-65).
ADAMs are expressed in a wide range of animal species, tissues and cell types and have been implicated in sperm-egg fusion, spermatogenesis, neutrophil infiltration, platelet aggregation, neurogenesis and cachexia. In addition, ADAMs have been shown to be involved in proteolysis of membrane anchored cytokines, growth factors and their receptors (Wolfsberg et al., J. Cell. Biol., 1995, 131, 275-8; Yamamoto et al., Immunol. Today, 1999, 20, 278-84).
ADAM10 (also known as MADM, HuAD10 and kuzbanian) was originally isolated from bovine brain and shown to act as a metalloprotease involved in the degradation of myelin basic protein (Howard et al., Biochem. J., 1996, 317, 45-50). This same enzyme was also shown to act as a type IV collagenase in the bovine kidney where it was shown to cleave a human placental basement membrane collagen preparation (Millichip et al., Biochem. Biophys. Res. Commun., 1998, 245, 594-8). In human cells, ADAM10 was first identified in THP-1 membrane extracts. Here it was shown to cleave a peptide substrate of tumor necrosis factor (TNF) alpha (Rosendahl et al., J. Biol. Chem., 1997, 272, 24588-93). Subsequently, it has been shown to act as a convertase with the ability to cleave both a recombinant pro-TNF-alpha and a synthetic peptide to the mature TNF-alpha protein (Lunn et al., FEBS Lett., 1997, 400, 333-5).
In the skeletal system, ADAM10 is expressed in specific regions of articular cartilage and metaphyseal bone of the neonatal rat tibia. In the bone and cartilage, it is expressed as two different transcripts of 4.5 kb and 7.5 kb by periosteal cells, osteoblasts and osteocytes at areas of active bone formation (McKie et al., Biochem. Biophys. Res. Commun., 1997, 230, 335-9; Dallas et al., Bone, 1999, 25, 9-15). These two isoforms are believed to be generated by the mechanisms of alternative splicing and/or alternative polyadenylation. In primary human osteoblasts and osteoblast cell lines, ADAM10 is localized in the trans-Golgi network and on the plasma membrane (Dallas et al., Bone, 1999, 25, 9-15).
More recently it has been demonstrated that the levels of ADAM10 mRNA are elevated in osteoarthritis tissues. These levels appear to be related to the degree of cartilage damage and/or degradation which suggests a role for ADAM10 in cartilage matrix destruction associated with osteoarthritis (Chubinskaya et al., J. Histochem. Cytochem., 1998, 46, 723-9).
ADAM10 has also been identified as an autoantigen in a patient with pulmonary fibrosis associated with dermatomyositis (Fujita et al., Ann. Rheum. Dis., 1999, 58, 770-2). Pulmonary fibrosis is an inflammatory disease of the lung.
In studies of cell lines from hematologic malignancies such as leukemia, erythroleukemia, lymphoma, and myeloma, ADAM10 expression was shown to be significantly elevated (Wu et al., Biochem. Biophys. Res. Commun., 1997, 235, 437-42). Here again, two transcripts were observed by Northern blot analysis.
The pharmacological modulation of ADAM10 activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions such as connective tissue disorders, inflammation and hematologic malignancies.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of ADAM10 and to date, strategies aimed at investigating ADAM10 function have involved the use of antibodies to localize ADAM10 (Millichip et al., Biochem. Biophys. Res. Commun., 1998, 245, 594-8). These antibodies were not shown to inhibit ADAM10 function and/or activity. Consequently, there remains a long felt need for agents capable of effectively inhibiting ADAM10 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of ADAM10 expression.
The present invention provides compositions and methods for modulating ADAM10 expression, including modulation of the alternatively spliced form of ADAM10.